Use of a methylol-containing compound to treat tumors

ABSTRACT

The invention provides a method of inhibiting tumor growth in a mammal, by administering to the mammal composition containing methylol-containing compound. The composition is administered to directly contact a tumor cell at a dose sufficient to induce cell death by apoptosis.

RELATED APPLICATION

This application claims the benefit from provisional application Ser.No. 60/169,122, which was filed on Dec. 6, 1999, provisional applicationSer. No. 60/169,127, which was filed on Dec. 6, 1999, and provisionalapplication Ser. No. 60/169,128, which was filed on Dec. 6, 1999.

BACKGROUND OF THE INVENTION

The invention relates to cancer therapy.

Despite advances in the identification of chemotherapeutic agents forinhibiting the growth of cancer cell, cancer remains a formidabledisease with a high mortality rate. A significant problem ofchemotherapeutic agents is low specificity. Many anticancer agents donot adequately distinguish normal cells from cancer cells. As a result,they often carry undesirable serious side effects.

SUMMARY OF THE INVENTION

The invention provides a method of inhibiting tumor growth in a mammalwith few or no deleterious side effects. The method is carried out byadministering to the mammal composition containing an activemethylol-containing compound. The compound is administered to directlycontact a tumor cell at a dose sufficient to induce cell death byapoptosis. Preferably the compound is administered in a manner and at adose which preferentially induces apoptotic death compared to necroticdeath. By “methylol containing compound” is meant a compound whichcontains or is capable of producing a methylol molecule in an aqueousenvironment. By “methylol containing compound” is meant a compound whichcontains or is capable of producing a methylol molecule in an aqueousenvironment. An aqueous environment includes conditions encounteredfollowing administration to a mammal, i.e., under physiologicalconditions. For example, a molecule of taurolidine produces threemethylol molecules, whereas a molecule of taurultam produces onemethylol species.

A method of treating an autologous tumor, e.g., a tumor of the centralnervous system (CNS), is carried out by administering to a mammal, e.g.,a human patient, a methylol-containing compound. The compound isadministered systemically, e.g., orally or intravenously, or infuseddirectly into the brain or cerebrospinal fluid. An erodible orresorbable solid matrix such as a wafer or sponge is implanted directlyinto brain tissue. Preferably, the tumor is a glioma, astrocytoma,neuroblastoma, or CNS metastasis from a non-CNS primary tumor.

The methylol-containing compound is characterized as having a R—CH₂—OHgroup in which R is an alkyl, aryl or hetero group. The invention alsoincludes compounds capable of producing or being converted into acompound containing a R—CH₂—OH structure. Preferably the compound istaurolidine, taurultam, or a derivative thereof (Tables 1, 2). R is analkyl, aryl, hydrogen or hetero group or atom.

Alternatively, the compound is a taurinamide derivative, e.g., acompound shown in Table 3; or a urea derivative, e.g., a compound shownin Table 4.

TABLE 1 Taurolidine Derivatives

R = alkyl, aryl, hydrogen, or hetero group or atom

TABLE 2 Taurultam Derivatives

R = alkyl, aryl, hydrogen, or hetero group or atom

TABLE 3 Taurinamide Derivatives

R = alkyl, aryl, hydrogen, or hetero group or atom

TABLE 4 Urea Derivatives

R = alkyl, aryl, hydrogen, or hetero group or atomOther methylol-containing compounds suitable for inducing apoptoticdeath of cancer cells include but are not limited to1,3,-dimethylol-5,5-dimethylhydantoin, hexamethylene tetramine, ornoxythiolin.By derivative of taurolidine or taurultam is meant a sulfonamidecompound which possesses at least 10% of the neoplastic activity oftaurolidine or taurultam, respectively. A sulfonamide compound is onehaving a R₂N—SO₂R′ formula. Derivatives of the compounds describedherein may differ structurally from a reference compound, e.g.,taurolidine or taurultam, but preferably retain at least 50% of thebiological activity, e.g., induction of apoptotic cell death, of thereference compound. Preferably, a derivative has at least 75%, 85%, 95%,99% or 100% of the biological activity of the reference compound. Insome cases, the biological activity of the derivative may exceed thelevel of activity of the reference compound. Derivatives may alsopossess characteristics or activities not possessed by the referencecompound For example, a derivative may have reduced toxicity, prolongedclinical half-life, or improved ability to cross the blood-brainbarrier.

The methylol-containing compound is administered alone or in combinationwith a second antineoplastic agent. Preferably, the coadministered agentkills tumors cells by a mechanism other than apoptosis. For example, anantimetabolite, a purine or pyrimidine analogue, an alkylating agent,crosslinking agent (e.g., a platinum compound), and intercalating agent,and/or an antibiotic is administered in a combination therapy regimen.The coadministered drug is given before, after, or simultaneously withthe methylol-containing agent. For example, taurolidine enhances theeffect of another drug or radiation therapy by increasing the number ofcertain types of cancer cells in “S” phase. An advantage of such acombination therapy approach is that a lower concentration of the secondneoplastic is required to achieve tumor cell killing.

The invention also includes treating a drug resistant tumor, e.g., amultiple drug resistant (MDR) tumor, in a mammal by administering to themammal a methylol-containing compound. The tumor to be treated is acarcinoma or sarcoma. The drug resistant tumor is selected from thegroup consisting of a solid tumor, a non-solid tumor, and a lymphoma.For example, the drug resistant tumor is a breast cancer, ovariancancer, colon cancer, prostate cancer, pancreatic cancer, CNS cancer,liver cancer, lung cancer, urinary bladder cancer, lymphoma, leukemia,or sarcoma.

Any neoplastic cell can be treated using the methods described herein.Preferably, the methylol-containing compound, e.g., taurolidine,taurultam, or a derivative thereof, is administered in a manner whichallows direct contact of the surface of the tumor cell with themethylol-containing compound. The compound itself or a methylol moleculeproduced by the compound binds to a component, e.g., a cell surfacepolypeptide ligand or other cell surface moiety to initiate anintracellular signal transduction cascade culminating with cell death byapoptosis. Tumors to be treated include but are not limited to leukemia,lymphoma, breast cancer, ovarian cancer, colon cancer, prostate cancer,pancreatic cancer, CNS cancer, liver cancer, urinary bladder cancer,sarcoma, and melanoma. For example, bladder cancer is treated byinflating the bladder with a solution containing a methylol-containingcompound, and skin cancers such as basal cell carcinomas or squamouscell carcinomas are treated by applying a methylol-containing compoundformulated as a film, cream, or ointment, directly to the affected skinarea. For treatment of primary liver cancers or liver metastases, thecompounds are infused into the hepatic artery, portal vein, or otherblood vessel of the liver. Alternatively, slow release of the compoundto any tissue is accomplished by implanting a drug loaded matrix indirect contact or adjacent to the tumor site.

To purge a mixed population of cells, e.g., a patient derived sample ofbone marrow cells or peripheral blood cells, of contaminating cancercells, the bone marrow cells or peripheral blood cells are cultured inthe presence of a methylol-containing compound such as taurolidine ortaurultam. The ex vivo treated cells are then washed and expanded inculture or infused into a mammalian recipient. e.g., the individual fromwhich the cells were derived or another mammalian recipient. The numberof tumor cells in the mixed population is reduced by at least one,preferably at least two, more preferably at least three, more preferablyat least four, and most preferably at least five log units, aftertreatment.

The methylol-containing compounds are formulated for administration todirectly contact cancer cells, e.g., in the form of an aqueous solution.Formulations include a therapeutic film-forming composition containingor coated with a methylol-containing compound as well as ointments,pastes, sprays, patches, creams, gels, sponges, and foams.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the structure of Taurolidine and its majorbreakdown products or metabolites (taurultam, taurinamide and taurine).Upon breakdown, each molecule of Taurolidine generates 3 methylolcontaining moieties implicated the antibiotic and anti-endotoxinactivities of taurolidine.

FIG. 2 is a bar graph showing the effect of a 48 h exposure toTaurolidine on the appearance of DNA debris in PA-1, SKOV-3 and NIH-3T3cells. Three×10⁵ cells were seeded in plastic tissue culture flasks.Twenty-four hours later, Taurolidine was added to achieve finalconcentrations of 25 μM, 50 μM or 100 μM. Control cultures received anappropriate volume of Kollidine-17P. After a 48 h period of Taurolidineexposure, cells were harvested and stained with propidium iodide. Thepercentage of DNA debris in the sub-G₀/G₁ region was assessed usingcytofluorometric techniques. Each bar represents the mean (±SE) of threedeterminations. **p≦0.01, ***p≦0.001, ****p≦0.0001

FIG. 3 is a bar graph showing the effect of a 24 h exposure toTaurolidine on membrane phosphotidylserine externalization in PA-1,SKOV-3 and NIH-3T3 cells. Three×10⁵ cells were seeded in plastic tissueculture flasks. Twenty-four hours later, Taurolidine was added toachieve final concentrations of 25 μM, 50 μM or 100 μM. Control culturesreceived an appropriate volume of Kollidine-17P. After an additional 24h, cells were harvested and phosphotidylserine externalizationdetermined by assessing Annexin-V FITC binding using cytofluorometrictechniques. Each bar represents the mean±SE of four determinations.**p≦0.01, ***p≦0.001

FIG. 4 is a photograph of showing the results of a Western-blot analysisof the effect of a 24 h exposure to 50 or 100 μM Taurolidine on PARPexpression and the appearance of a major PARP cleavage product in PA-1,SKOV-3 and NIH-3T3 cells. Two×10⁶ cells were seeded in 150 cm² tissueculture flasks. Twenty-four hours later, Taurolidine was added atconcentrations of 50 μM or 100 μM. After an additional twenty-fourhours, cells were harvested, cell number determined, and aliquotsderived from equal cell numbers generated from each exposure condition.Total proteins from these whole cell lysates were separated by SDS-PAGEand transferred to nitrocellulose filters. Filters were thenimmunoblotted to detect intact PARP protein and cleavage fragments byusing the clone C-2-10 mouse monoclonal anti-PARP antibody (ZymedLaboratories, San Francisco, Calif.). The resulting protein-antibodycomplexes were visualized by chemiluminescence techniques.

FIG. 5 is a bar graph showing the effect of delayed administration of asingle 3 d i.p. bolus injection regimen of Taurolidine (20mg/mouse/injection) on the occurrence of i.p. human tumor xenografts infemale nude mice following the i.p. administration of 5×10⁶ SKOV-3 humanovarian tumor cells. Taurolidine therapy was initiated on the day oftumor cell inoculation or up to 5 d thereafter. Fourteen days followingthe final Taurolidine injection, mice in all groups were sacrificed andthe peritoneal cavity examined for the presence of tumors. Eachexperimental was repeated three times and the pooled number of animalsin each group ranged from 15-21.

FIG. 6 is a bar graph showing the effect of delayed administration of asingle 3 d i.p. bolus injection regimen of Taurolidine (20mg/mouse/injection) on the weight of i.p. human tumor xenografts infemale nude mice following the i.p. administration of 5×10⁶ SKOV-3 humanovarian tumor cells. Taurolidine therapy was initiated on the day oftumor cell inoculation or up to 5 d thereafter. Fourteen days followingthe final Taurolidine injection, mice in all groups were sacrificed,i.p. ovarian tumor xenografts removed and tumor weighed. Each experimentwas repeated three times and the pooled number of animals in each groupranged from 15-21. Each bar represents the mean (±SE) tumor weight of15-21 animals. ***p≦0.001, ****p≦0.0001.

DETAILED DESCRIPTION

Taurolidine and taurultam were found to be safe and effectiveantineoplastic agents which preferentially induce apoptotic death incancer cells. Compounds which function as methylol donors can be used toinduce apoptotic death of tumor cells. A functional group (methylol) canbe incorporated into a variety of compounds which then act as methylolcarriers.

Methylol-containing Compounds

Taurolidine was found to be selectively toxic to cancer cells withoutkilling normal (i.e., noncancerous) cells. Upon administration to asubject, taurolidine, in an aqueous environment exists in equilibriumwith several methylol-containing or producing species. Hydrolysis oftaurolidine in vivo leads to methylol taurultam and taurultam inequilibrium. Administration of taurolidine to a mammal also producesN-methylol-taurinamide and taurultam. The active antineoplastic agent isa methylol-containing compound.

Upon hydrolysis of taurolidine, in aqueous solutions, one methylol groupis formed from the bridgehead methylene between the rings astaurultam-methylol. Taurultam is further transferred via methyloltaurinamide to taurine. Thus, three active methylol molecules areproduced per molecule of taurolidine following administration to amammal. One active methylol molecule is produced per molecule oftaurultam, and two active methylol molecules are produced fromtaurultam-methylol. Each methylol group has high affinity for and bindsselectively to a moiety on the surface of a cancer cell (e.g., aphosphatidyl serine receptor) and induces apoptosis in that cell, whichin turn leads to cytotoxicity. Cellular internalization of amethylol-containing compound or a methylol molecule may not be necessaryfor induction of apoptotic death of a cancer cell.

Cytotoxicity or cell death may occur by either necrosis or apoptosis.Necrosis, which is not genetically controlled, is usually the result ofphysical or chemical injury. Apoptosis is genetically controlled and isa cellular response to a specific stimuli, e.g., a cellsurface-generated signal. Necrosis involves the destruction ofcytoplasmic organelles and a loss of plasma membrane integrity, whereascells undergoing apoptosis exhibit cell shrinkage, membrane blebbing,chromatin condensation and fragmentation. After the DNA damage in thecaspase enzyme pathway, there are a series of events which occur thatinvolve calcium activation and calpain enzymes which further leads toother cellular changes and regulation of cytoplasmic enzymes.

A major difference between necrosis and apoptosis in vivo is theelimination of the apoptotic cell before an inflammatory response ismounted. In contrast to apoptosis of cells, necrosis of cells causesinflammation. Thus, induction of cytotoxicity of cancer cells byapoptosis offers considerable advantages over induction of cell death byconvention chemotherapeutic agents because apoptotic death is associatedwith minimal damage to surrounding cells or tissue. Unlike manyconventional chemotherapeutic agents, methylol-containing agents such astaurolidine or taurultam administered according to the invention arecytotoxic agents which induce apoptosis of cancer cells (but not normalnoncancerous cells) to safely reduce the tumor burden in a mammalsuffering from cancer.

Functional Characterization of Taurolidine

Taurolidine (Taurolin™) is chemically identified asBis-(1,1-dioxoperhydro-1,2,4-thiadiazinyl-4)methane (FIG. 1). It is arelatively small dimeric molecule with a molecular weight of 284 (Knightet al., 1983, J. Pharm. Sci 72:705-707)). Early assessment of itsantibiotic activity revealed that it possessed bactericidal activityagainst a broad spectrum of aerobic and anaerobic bacterial strains. Theminimum concentration required to inhibit bacterial cell growth (MIC)ranged from 0.01 to 1 mg/ml, depending on the bacterial strainevaluated. Early studies also revealed that Taurolidine possessedactivity against clinically relevant fungi. The concentration requiredto exert antifungal activity is approximately equivalent to thatrequired to produce its antibacterial activity.

The antibiotic activity of Taurolidine depends upon a chemical reactionsecondary to the generation of active methylol groups formed upon thedecomposition of the parent Taurolidine molecule (FIG. 1). Biochemicaland morphological studies revealed that Taurolidine-derived methylolgroup containing moieties appeared to react with bacterial cell wallcomponents. The result of this chemical reaction is that exposure tothis agent significantly inhibits the ability of microorganisms toadhere to biological surfaces, such as epithelial cells. Exposure toTaurolidine disrupted the structure, and reduced the number, ofbacterial cell fimbriae, apparently a reflection of the agglutination ofthese structures. Modification of these surface structures is thought tobe responsible for the ability of Taurolidine to disrupt bacterial celladhesion. In addition to this direct effect on bacterial cell wallcomponents, Taurolidine also possesses anti-endotoxin activity byreducing tumor necrosis factor alpha (TNF-α) synthesis and activity.Taurolidine also reduces the extent and severity of postoperativeperitoneal adhesions and has been administered clinically, by lavage,after abdominal surgery to reduce post-operative infections andadhesions as well as to treat peritonitis.

Taurolidine is a synthetic broad-spectrum antibiotic that also possessesantifungal activity. Mechanistically, it reacts with bacterial cellmembrane components to prevent the adhesion of bacterial cells toepithelial cell surfaces. Reflecting the key role of cell adhesion inthe growth and development of human solid tumors, studies were initiatedto assess the cytotoxic activity of this agent against the growth of apanel consisting of 12 selected human and murine tumor cell lines.Assessment of the growth inhibitory activity of a 3 d Taurolidineexposure revealed that this agent inhibited the growth of all cell linesevaluated with IC₅₀s ranging from 9.6-34.2 μM. Studies to identify theunderlying mechanism(s) responsible for this effect were conducted inNIH-3T3 murine fibroblasts and the PA-1 and SKOV-3 human ovarian tumorcell lines. Initial studies assessed the effect of a 48 h exposure toTaurolidine on cell cycle distribution. The results of this analysisrevealed that while Taurolidine had little effect on the cell cycle inPA-1 cells, in SKOV-3 cells it reduced the percentage of cells in theG₀/G₁-phase and increased the percentage of cells in both S and G₂/M. Inthese human tumor cell lines, Taurolidine exposure significantlyincreased DNA debris in the sub-G₀/G₁ region, an effect consistent withan induction of apoptosis. In contrast, in NIH-3T3 cells, Taurolidineincreased the percentage of cells in S-phase, decreased the percentageof cells in G₀/G₁, and did not increase DNA debris in sub-G₀/G₁ region.Further studies of the relationship between Taurolidine exposure andtumor cell apoptosis assessed phosphotidylserine externalizationfollowing a 24 h exposure to Taurolidine, using Annexin-V binding as acell surface marker. These studies revealed that Taurolidine increasedthe percentage of Annexin-V positive cells by 4- and 3-fold in PA-1 andSKOV-3 cells, respectively. In contrast, in NIH-3T3 cells, Taurolidineexposure slightly increased (˜5%) Annexin-V binding. Complementarystudies determined if a 48 h exposure to either 50 or 100 μM Taurolidineaffected PARP cleavage in these cell models and revealed thatTaurolidine induced PARP cleavage in both PA-1 and SKOV-3 cells. Intotal, these in vitro results reveal that Taurolidine possesses tumorcell cytotoxic activity that correlates with its ability to specificallyinduce apoptosis. Finally, murine-based studies were conducted to assessthe antineoplastic activity of this agent. Initial studies assessed thetoxicity of 3 consecutive daily i.p. bolus injections of Taurolidine, atdoses ranging from 5 mg/inj/mouse-30 mg/inj/mouse. The 20 mg/inj doseproduced ˜10% mortality and was identified as the MTD in this model.Administration of this Taurolidine regimen to nude mice bearing i.p.human ovarian tumor xenografts resulted in a significant inhibition ofboth tumor formation and growth.

The invention is based on the discovery that, in addition to theactivities discussed above, taurolidine selectively and reliablyinhibits tumor cell growth and selectively kills tumor cells by inducingapoptosis. Taurolidine has now been found to kill at least 28 differenthuman tumor cell lines including ovarian, breast, brain, colon,prostate, urinary bladder and lung tumors, as well as melanomas,mesotheliomas, laryngeal carcinomas, leukemias, and lymphomas. Inaddition, multiple-drug resistant glioma clls and myelodysplasticsyndrome cells (a precancerous cell type) were killed by taurolidine.Inhibition of tumor growth and induction of tumor cell death occur attaurolidine concentrations significantly lower than those required forantibiotic activity. For example, for antineoplastic applications,taurolidine is administered at a dose that is at least 10% less,preferably at least 20% less, more preferably at least 50% less, and upto one log unit less than the dose required for antibacterial orantiadhesive activity.

Taurolidine is toxic to tumor cells (but not normal non-tumor cells)regardless of the tumor origin. Apoptosis of tumor cells s induced afteran incubation with Taurolidine for as little as one hour in culture.

Taurolidine and metabolites thereof are also useful in combinationtherapy. The data indicate that taurolidine is useful to enhance thecytotoxicity of other chemotherapeutic agents and/or radiation therapyby inducing certain types of cancer cells to enter “S” phase.

Taurolidine and Angiogenesis

Patients with metastatic colon cancer were treated with taurolidine andseveral factors controlling angiogenesis were measured. Four factorscontrolling the growth of blood vessels (tissue necrosis factor (TNF);interleukins 1, 6, and 10; vascular endothelial growth factor (VEGF);and tumor growth factor-β (TGF)) were found to be decreased intaurolidine-treated subjects compared to subjects receiving placebo.These data indicate that taurolidine has anti-angiogenesis activity andis useful to inhibit tumor growth by decreasing new blood vesselformation.

Therapeutic Administration

An effective amount of a methylol-containing compound is preferably fromabout 0.1 mg/kg to about 150 mg/kg. However, due to the low toxicity oftaurolidine and taurultam compounds, higher doses may be administeredwithout deleterious side effects. A dose effective to induce apoptosisof cancer cells is an order of magnitude less than doses administeredfor antiseptic, antibacterial, antitoxic, or anti-adhesion purposes. Anapoptotic dose of taurolidine or taurultam effective to induce apoptosis(e.g., 0.5 μg/dl) is also significantly less than doses previouslysuggested (e.g., 150-450 mg/kg) as potentially being useful in thetreatment of certain cancers. Effective doses vary, as recognized bythose skilled in the art, depending on route of administration,excipient usage, and coadministration with other therapeutic treatmentsincluding use of other antitumor agents (e.g., an antimetabolite, apurine or pyrimidine analogue, an alkylating agent crosslinking agent,intercalating agent, or an antibiotic.) and radiation therapy.

A therapeutic regimen is carried out by identifying a mammal, e.g., ahuman patient suffering from (or at risk of developing) a cancer ormetastases using standard methods. For example, taurolidine or taurultamis administered to an individual diagnosed with a cancer (e.g., acutemyeloid leukemia) or an individual diagnosed with a precancerouscondition (e.g., myelodysplasia which may progress to acute myeloidleukemia). The pharmaceutical compound is to administered to such anindividual using methods known in the art. Preferably, the compound isadministered orally, topically or parenterally, e.g., subcutaneously,intraperitoneally, intramuscularly, and intravenously. For example,ovarian cancer may be treated by intraperitoneal lavage using apharmaceutically-acceptable solution of taurolidine or taurultam. Thecompound is administered prophylactically, after the detection of arecurring tumor, or at the time of surgery. The compound may beformulated as a component of a cocktail of chemotherapeutic drugs) totreat a primary ovarian cancer or to prevent recurring tumors. Examplesof formulations suitable for parenteral administration include aqueoussolutions of the active agent in an isotonic saline solution, a 5%glucose solution, or another standard pharmaceutically acceptableexcipient. Standard solubilizing agents such as PVP or cyclodextrins arealso utilized as pharmaceutical excipients for delivery of thetherapeutic compounds.

A methylol-containing compound is formulated into compositions for otherroutes of administration utilizing conventional methods. For example, itcan be formulated in a capsule or a tablet for oral administration.Capsules may contain any standard pharmaceutically acceptable materialssuch as gelatin or cellulose. Tablets may be formulated in accordancewith conventional procedures by compressing mixtures of amethylol-containing compound with a solid carrier and a lubricant.Examples of solid carriers include starch and sugar bentonite. Thecompound is administered in the form of a hard shell tablet or a capsulecontaining a binder, e.g., lactose or mannitol, a conventional filler,and a tableting agent. Other formulations include an ointment, paste,spray, patch, cream, gel, resorbable sponge, or foam. Such formulationsare produced using methods well known in the art.

Methylol-containing compounds such as taurolidine or taurultam areeffective upon direct contact of the compound with the cancer cell.Accordingly, the compound is administered topically. For example, totreat urinary bladder carcinoma, the compound is administered to thebladder using methods well known in the art, e.g., using a catheter toinflate the bladder with a solution containing the methylol-containingcompound for at least ten minutes. For example, the bladder is instilledwith a solution of taurolidine or taurultam, and the solution allowed toremain in the bladder for 30 minutes to 2 hours. For treatment of skinmalignancies such as basal cell carcinomas, a cream or ointment isapplied to the area of skin affected by the tumor. Tumor cells in theliver (e.g., a primary tumor or liver metastases originating fromprimary tumor elsewhere in the body such as the colon or breast) aretreated by infusing into the liver vasculature a solution containing amethylol-containing agent. Alternatively, the compounds are administeredby implanting (either directly into an organ such as the liver orsubcutaneously) a solid or resorbable matrix which slowly releases thecompound into adjacent and surrounding tissues of the subject.Implantation of a drug-loaded matrix directly into the liver effectivelydestroys tumor cells in the liver, while healthy liver tissue rapidlydetoxifies any residual chemotherapeutic agent.

For treatment of cancers of the CNS such as glioblastomas, the compoundis systemically administered or locally administered directly into CNStissue. The compound is administered intravenously or intrathecally(i.e., by direct infusion into the cerebrospinal fluid). For localadministration, a compound-impregnated wafer or resorbable sponge isplaced in direct contact with CNS tissue. A biodegradable polymerimplant such as a GLIADEL™ wafer is placed at the tumor site, e.g.,after surgical removal of a tumor mass. A biodegradable polymer such asa polyanhydride matrix, e.g., a copolymer of poly (carboxy phenoxypropane):sebacic acid in a 20:80 molar ratio, is mixed with atherapeutic agent, e.g., taurolidine or taurultam and shaped into adesired form. Alternatively, an aqueous solution or microsphereformulation of the therapeutic agent is sprayed onto the surface of thewafer prior to implantation. The compound or mixture of compounds isslowly released in vivo by diffusion of the drug from the wafer anderosion of the polymer matrix. A methylol-containing compound such astaurolidine or taurultam may be coadministered with otherchemotherapeutic agents such as carmustine (BCNU).

Alternatively, the compound is infused into the brain or cerebrospinalfluid using known methods. For example, a burr hole ring with a catheterfor use as an injection port is positioned to engage the skull at a burrhole drilled into the skull. A fluid reservoir connected to the catheteris accessed by a needle or stylet inserted through a septum positionedover the top of the burr hole ring. A catheter assembly (e.g., anassembly described in U.S. Pat. No. 5,954,687) provides a fluid flowpath suitable for the transfer of fluids to or from selected locationat, near or within the brain to allow administration of the drug over aperiod of time.

The compounds are also used to purge a sample of bone marrow cells ofcancer cells which may contaminate the sample. Bone marrow cells arederived from a mammalian donor using standard methods. The cells aretreated by contacting them with a methylol-containing compound in vitroto eliminate contaminating tumor cells. After washing the treated cells,the bone marrow cell preparation is administered to a mammalianrecipient to reconstitute the immune system of the recipient.

Similarly, a population of peripheral blood mononuclear cells is purgedof tumor cells. Peripheral blood may be used as a source of stem cells,e.g., hematopoetic stem cells, for repopulating the immune system of acancer patient following chemotherapy or radiation therapy. In somecases (e.g., patients with a myeloma or breast cancer), using peripheralblood as a source of stem cells is preferable to using bone marrowbecause the peripheral blood may be less contaminated with tumor cells.Peripheral blood mononuclear cells are obtained from an individual usingstandard methods, e.g., venipuncture or plasmapheresis. The cells aretreated with a methylol-containing compound such as taurolidine,taurultam, or a derivative thereof, in vitro to kill contaminating tumorcells. The cells are washed and infused into a recipient individual.Optionally, the cells are cultured to expand a desired cell type.

Cytotoxicity of Methylol-containing Compounds

The cytotoxic activity of taurolidine was evaluated in vitro against thegrowth of a variety of human cancer cell lines as well as “normal” NIH3T3 fibroblasts and found to induce apoptotic cytotoxicity. Theneoplastic cell lines used in the survey were standard tumor cell lines,e.g., PA1 human ovarian cell line, SKOV3 human ovarian cell line, HT29human colon tumor cell line, DU145 human prostate tumor cell line, U251human glioblastoma cell line, U251-MDR human glioblastoma cell linetransfected with DNA encoding MDR, T98G human glioblastoma cell line,SP-1 human leukemia cell line, and Daudi human leukemia cell line.

The data indicated that taurolidine inhibited human cancer cell growth.Surprisingly, the concentration of taurolidine required to inhibit tumorcell growth after a 3-day exposure to the compound (IC₅₀) wasapproximately 12.5 μM-50 μM. This concentration is at least 1000-foldlower than concentrations used to inhibit bacterial cell growth.

Taurolidine and cancer cells were added to flasks simultaneously, andcell growth was assessed 3 days later. Parallel studies were carried outto assess whether disruption of cell adhesion played a role in thecytotoxic activity. Assays were carried out to assess the ability oftaurolidine to inhibit the growth of human ovarian tumor cells afterthey were established and growing in vitro as discrete colonies. Thedata revealed that a 24-hour exposure to 50 μM taurolidine produced asignificant cytotoxic effect against the growth of established tumorcells. The data indicated that the cytotoxic/cytostatic activity oftaurolidine is not due to inhibition of tumor cell adhesion.

The mechanism by which taurolidine produces cytoxicity was evaluated.Cell cycle kinetics and cell cycle distribution of tumor cells wereexamined after a 24-hour exposure to taurolidine. The results revealedthat in both PA1 and 3T3 cells, taurolidine exposure disrupted cellcycle kinetics and significantly reduced the percentage of cells in boththe S- and G2/M-phases. Exposure of PA1 human ovarian cells to thisregimen of taurolidine also induced a high degree of DNA fragmentationindicating the induction of apoptosis. This DNA fragmentation was notobserved in normal 3T3 cells.

To further evaluate the possibility that exposure to 50 μM taurolidinewas capable of specifically inducing apoptosis in human ovarian tumorcells but not normal fibroblasts, studies were undertaken to evaluateDNA fragmentation as a function of taurolidine exposure by usingagarose-gel electrophoresis. The results confirmed that, in ovariantumor cells, exposure to taurolidine resulted in overt DNA fragmentationwhich was not apparent in 3T3 cells exposed to an identical taurolidineregimen.

The cytotoxic activity of taurultam was evaluated in vitro using thesame human cancer cell lines as described above. The data indicated thattaurultam induced apoptotic death of cancer cells but not normal controlcells in the same manner as taurolidine. The cytotoxic activity oftaurultam was approximately 75% of the activity observed withtaurolidine. These data confirm that a class of compounds defined by theability to release a methylol moiety exerts a potent and specificcytotoxic effect on cancer cells. The data also indicate that thecytotoxic effect is directly proportionate to the number of methylolmolecules liberated per molecule of methylol-containing compound

Apoptotic death is distinguished from death by other mechanisms usingmethods known in the art. Another early reflection of the induction ofapoptosis is the cleavage of the protein poly (ADP-ribose) polymerase(PARP) by cellular caspases. Western-blot based studies were carried outto determine if exposure to taurolidine resulted in PARP cleavage. Theresults revealed that PARP cleavage was not evident in 3T3 cells whenexposure to the same taurolidine regimen. Apoptosis is also detectedusing known methods such as determination of caspase activation,bax/bcl12 ratios and fas and fas-I interactions. Other methods ofdistinguishing between apoptosis and necrosis (e.g., afluorescence-based method described in U.S. Pat. No. 5,976,822) are usedto determine the mechanism of death or the dose at which amethylol-containing compound induces apoptosis compared to necrosis.

The antitumor activity of a compound is also evaluated using a standardMTS calorimetric assay. Results obtained with various types of tumorcells (primary cells or cell lines) are compared with those obtained byusing normal cells. Viability of the cells in each cell line isestimated by measuring the cellular conversion of a tetrazolium saltafter incubating the cells in a solution containing a test compound in a96 well plate. IC₅₀ values obtained using the identical test compound onnormal cells and cells of a particular tumor cell line are compared andtheir ratio (IC₅₀ normal cell/IC₅₀ cancer cell) indicates the cancerselectivity of the test compound. An increase in the IC₅₀ normalcell/IC₅₀ cancer cell ratio reflects a higher selectivity of the testcompound to kill the cancer cell.

Antitumor activity of a compound is also evaluated in vivo using, e.g.,a tumor xenograft regression assay. For example, animals bearingestablished tumors are treated with a test compound for a three-weekperiod. The growth of the tumors and the general health of the animalare monitored during the three-week treatment and for two more weeksafter treatment to determine if tumor regrowth occurs. Theantineoplastic activity of taurolidine is determined in athymic (nude)mice bearing advanced and/or metastatic xenografts. Single and multipledose regimens of taurolidine are evaluated in athymic (nude) mice. Uponidentification of dose regiments, antineoplastic activity is assessed inathymic (nude) mice bearing xenografts of human cancer cells, e.g.,ovarian, prostate, colon, pancreatic, breast and glioma tumors.

Treatment of Leukemias and Lymphomas

The compounds described herein are particularly effective in killingtumor cells which are not anchorage-dependent such as leukemias orlymphomas. The cytotoxic effect is not due to inhibition of celladhesion.

Two different non-anchorage-dependent tumor cell lines (a humanBurkitt's lymphoma cell line, and a Daudi cell line), and precancerouscell line (a human myelodysplastic cell line) were grown in suspensionculture. After exposing the tumor cells to 10-20 μM of taurolidine for72 hours, 50% of the cells died. Similar results were observed afterexposure of the myelodysplastic cells to taurolidine. These resultsindicate that taurolidine is useful to treat non-anchorage-dependenttumor cell types such as lymphomas or leukemias. The results alsoindicate that precancerous cells such as myelodysplastic cells areeffectively killed by the compounds described herein and thatindividuals diagnosed with myelodysplasia (which may develop into anacute myeloid leukemia) may be effectively treated using taurolidine orother methylol-containing compounds described herein.

Treatment of Ovarian Cancer

Over 80% of patients diagnosed with ovarian cancer experience recurrenttumors after therapeutic intervention for the primary tumor. Even a 5%response rate, e.g., a 5% reduction in tumor growth, would confer aclinical benefit. Response rate is defined as a reduction in tumor sizeor in the number of metastatic foci. For example, a reduction in tumorsize is determined by detecting a decrease in the size of the largestneoplastic lesion, e.g., by sonograrn or by measurement using a caliper.

A standard mouse model of ovarian cancer was used to study the effect oftaurolidine on recurrent ovarian cancer. Holland Sprague-Dawley micewere injected with 5×10⁶ tumor cells (e.g., SKOV3 human ovarian tumorcell line) to mimic a condition of advanced ovarian cancer. Taurolidinewas administered by intraperitoneal lavage 5 days later. Taurolidine wasadministered 3 times a day for 4 days at a dose of 30 mg/day. At least a75-80% reduction in tumor foci was observed. These data indicate thatadministration of taurolidine reduces ovarian tumor burden andrecurrence of tumors.

Treatment of Drug Resistant Tumors

Taurolidine was found to be particularly effective in killing tumorcells which are refractory to cytotoxicity by other knownchemotherapeutic agents. Glioblastoma cells were transfected with a geneencoding multiple drug resistance (MDR). The transfected cells were100-1000 times resistant to standard chemotherapeutic agents, e.g.,adriamycin. Untransfected glioblastoma cells cultured with a standarddose (e.g., 1 μM) of adriamycin were killed, but MDR-transfectedglioblastoma cells contacted with 1 μM of the drug were resistant.Significant cytotoxicity of the MDR-transfected glioblastoma cells wasobserved after contact with a methylol-containing compound (e.g.,taurolidine at a dose of 50 μM). These data indicate thatmethylol-containing compounds such as taurolidine exert their cytotoxicactivity via a mechanism that differs from that of standardchemotherapeutic agents. Accordingly, combination therapy in which amethylol-containing compound is administered before, after, or togetherwith another chemotherapeutic agent (e.g., an antimetabolite, atumor-specific monoclonal antibody, or anti-angiogenic agent) results inimproved clinical outcome of patients suffering from a malignantcondition characterized by a mixed population of tumor cells (e.g.,those which are killed by standard chemotherapeutic agents and thosewhich are MDR).

EXAMPLE 1 Cytotoxic and Mechanistic Evaluation of Antineoplastic Agents

Taurolidine was found to be active at inhibiting the growth of a varietyof human tumor cell lines in vitro. PA-1 and SKOV-3 human ovarian tumorcell lines and NIH-3T3 murine fibroblasts were used to determine themechanism of antitumor activity. The studies revealed that this effectwas associated with alterations in DNA structure, cell membranecomponents, and protein cleavage that were consistent with the inductionof apoptosis specifically in tumor cells. Antineoplastic evaluation ofTaurolidine in nude mice bearing intraperitoneal xenografts of humanovarian tumors demonstrated that this agent significantly inhibitedtumor development and growth in vivo.

To study neoplastic activity, Taurolidine was formulated as 2% solutionin 5% Kollidon 17PF. Standard cell culture growth media (e.g., Highglucose DMEM, RPMI 1640, McCoy's 5A, and F12K), trypsin, and fetalbovine serum (FBS) were all purchased from GIBCO/Life Technologies(Grand Island, N.Y.). Phosphotidylserine externalization by cells wasevaluated using the ApoAlert® Annexin-V/FITC assay kit was purchasedfrom Clontech (Palo Alto, Calif.). Reagents for SDS-PAGE were purchasedfrom BioRad Laboratories (Richmond, Calif.). A murine monoclonalantibody (clone C-2-10) to human PARP was purchased from ZymedLaboratories (San Francisco, Calif.). All other chemicals were purchasedfrom Sigma (St. Louis, Mo.).

Studies to assess the cytotoxic activity of Taurolidine were carried outusing a panel of human solid tumor cell lines as well as in NIH-3T3murine fibroblasts. Included in the tumor cell line panel were ovariantumor cells (PA-1 and SKOV-3), colon tumor cells (HCT-8, HCT-15 andHT-29), lung tumor cells (H-157, A-549 and H-596), prostate tumor cells(DU-145), glioma cells (U-251), and melanoma (MNT-1). The murinemelanoma B16F10 cell line was also tested. These cell lines readilyavailable, e.g., from the American Type Culture Collection (ATCC). Cellswere cultured in appropriate growth medium at 37° in a humidifiedincubator in an atmosphere of 5% CO₂. Under these growth conditions, thedoubling time of all cell lines was 20-28 h.

Studies to assess in vivo toxicity and therapeutic effectiveness werecarried out in 6-12 week old female homozygous athymic (Hsd:athymic nudenu/nu) mice obtained from Harlan (Indianapolis, Ind.).

To evaluate inhibition of cell growth, subconfluent cultures ofappropriate cell lines were harvested by trypsinization and resuspendedin media at a cell density of 1-5×10⁴ cells/ml. One ml of this cellsuspension was added to each well of a 12 well cell culture plate thatcontained 3 ml of appropriate media plus serum. Twenty-four hours later,Taurolidine was added to each well, in a volume of 40 μl, to achieve afinal concentration of 0.1-200 μM. Control wells received 40 μl of 5%Kollidon 17PF alone. Seventy-two hours later, all cells were harvestedby trypsinization and cell number determined electronically using aCoulter Model Z1 particle counter (Coulter Corp., Miami, Fla.) to assesscell growth inhibition. Each experiment was performed in duplicate andrepeated a minimum of three times.

For flow cytometry studies, 1×10⁶ PA-1, SKOV-3, or NIH-3T3 cells wereincubated for 24 h in appropriate media containing serum. Twenty-fourhours later, Taurolidine was added in a volume of 40 μl to achieve afinal concentration of 25, 50, or 100 μM. Control cultures for each cellline were incubated in media containing 40 μl of 5% Kollidon 17PF alone.Forty-eight hours later, all cells were harvested by trypsinization andprepared for cytofluorometric analysis by standard methods. For example,harvested cells were resuspended in ice cold phosphate-buffered salineat a final cell density of 2×10⁶ cells/ml. The cells then were stainedfor 30 min at room temperature in the dark with a solution of 0.05 mg/mlpropidium iodide, 0.6% Igepal, and 1% sodium citrate. Flow cytometry wasperformed by FACScan (Becton Dickinson, Plymouth, England) using theModFit LT program (Becton Dickinson). Statistical analysis was performedwith the Kruskal Wallis non-parametric ANOVA test followed by Dunn'smultiple comparisons test using Instat.

Cell membrane phosphotidylserine externalization, as a reflection of thepotential induction of apoptosis, was assessed by flow cytometry methodsusing the ApoAlert® Annexin-V/FITC assay kit. Briefly, 1×10⁶ cells wereincubated for 24 h in tissue culture medium containing serum.Thereafter, Taurolidine was added to achieve a final concentration of25, 50, or 100 μM. Control cultures received 5% Kollidon 17PF alone.Twenty-four hours later, all cells were harvested by trypsinization. Theharvested cells were resuspended in 200 μl of binding buffer and thenincubated for 5-15 min in a solution containing 1 μg/ml Annexin-V FITCat room temperature in the dark. The cells were then analyzed toquantitate Annexin-V binding by cytofluorometric techniques thatutilized FACScan using the ModFit LT program with statistical analysisas described above.

Western-blot analysis was used to assess of PARP cleavage. Two×10⁶ cellswere seeded into separate 75 cm² tissue culture flasks containing 20 mlof tissue culture media plus serum. Twenty-four hours later, Taurolidinewas added at concentrations of 50 μM or 100 μM. Twenty-four hours afterthe addition of Taurolidine, cells were harvested, cell numberdetermined, and aliquots containing an equal cell number were generatedfrom each exposure condition. Total proteins from whole cell lysatesgenerated from these aliquots were separated by SDS-PAGE andelectrotransferred to nitrocellulose filters. Filters were thenprocessed to detect intact PARP protein and cleavage fragments by usingthe clone C-2-10 mouse monoclonal anti-PARP antibody (ZymedLaboratories, San Francisco, Calif.). The resulting protein-antibodycomplexes were visualized by standard chemiluminescence techniques.

To evaluate Taurolidine-induced toxicity, mice were divided into groupsof 5-8 animals. Thereafter, all mice were weighed and therapy,consisting of a single i.p. bolus injection of Taurolidine on 3consecutive days, was initiated. The Taurolidine doses evaluated were 5,10, 15, 20, 25, and 30 mg/mouse/injection and, except for the 25 (1.25ml) and 30 mg/mouse (1.5 ml) injections, were administered in a volumeof 1 ml. Taurolidine for injection was diluted from the 2% Taurolidinesolution by the addition of 5% Kollidon 17PF. Control animals received 1ml injections of 5% Kollidon 17PF alone. Animals were examined daily andbody weight recorded twice weekly. A reduction in body weight of greaterthan 10% was considered significant. The maximally tolerated dose (MTD)was considered to be the dose which produced ˜10% mortality.

To evaluate therapeutic effectiveness, mice received a singleintraperitoneal injection of 5×10⁶ SKOV-3 cells in a volume of 0.5 ml.Immediately thereafter, mice were randomly divided into treatment groupsof 7 animals. Taurolidine therapy, consisting of a single i.p. bolusinjection of 20 mg of Taurolidine on 3 consecutive days, was initiatedeither immediately following tumor cell inoculation or at selected timeintervals after tumor cell inoculation (≦5 d). Control animals received1 ml injections of 5% Kollidon 17PF alone. Animals were examined dailyand body weight recorded twice weekly. Fourteen days following the lastTaurolidine injection, mice in all groups were sacrificed by CO₂asphyxiation, all i.p. tumor foci removed and tumor weighed determined.The mean tumor weight for each treatment group was calculated andstatistical analysis of differences in the mean tumor weight betweentreatment groups employed the Student's t-test. p-values of ≦0.05 wereconsidered significant.

Taurolidine Inhibits Tumor Cell Growth

The ability of Taurolidine to inhibit cell growth was assessed in apanel of human and murine neoplastic cell lines comprised of 13different lines representing 6 different tumor types. The results ofthis survey revealed that a 3 d exposure to Taurolidine inhibited cellgrowth in each cell line examined (Table 5).

The IC₅₀ of Taurolidine against the growth a selected human and murineneoplastic cell lines was evaluated as follows. Cells were seeded at adensity of 1-5×10⁴ cells in each well of a 6 well tissue culture flask.Twenty-four hours later, Taurolidine was added at concentrations rangingfrom 1-100 μM. After three days, cells were harvested by trypsinizationand cell number determined electronically. Cell growth inhibition wasdetermined by comparison to non-Taurolidine exposed control cultures.The IC₅₀ was calculated as the concentration required to inhibit cellnumber by 50%. Each IC₅₀ value represents the mean±SE of 4-8determinations.

TABLE 5 Tumor site of origin Cell line IC₅₀* (uM) Ovary PA-1 11.4 ± 1.8SKOV-3 31.6 ± 7.0 Prostate DU-145  9.8 ± 0.8 Brain U-251 20.1 ± 2.7Colon HT-29 18.6 ± 1.0 HCT-8 11.5 ± 0.5 HCT-15  9.6 ± 3.0 MelanomaB16-F10 30.1 ± 2.6 MNT-1 22.1 ± 2.1 Lung H-157 32.2 ± 5.6 A-549 26.8 ±7.2 H-596 34.2 Murine fibroblasts NIH-3T3 11.9 ± 1.8

Surprisingly, the observed IC₅₀s for each cell line were remarkablysimilar and varied over the relatively narrow range of ˜10 μM (PA-1,DU-145, HCT-8, HCT-15, B16F10, and NIH-3T3) to ˜35 μM (H-596).

The studies assessed the effect of Taurolidine on tumor cellproliferation. Inhibition of proliferation could reflect either growtharrest or cell death. Therefore, studies were next focused to identifythe mechanism(s) by which Taurolidine induced cell growth inhibition.These studies were carried out in the human ovarian tumor cell linesPA-1 and SKOV-3 and in NIH-3T3 murine fibroblasts. Studies employingconventional flow cytometry techniques assessed the effect of a 48 hexposure to Taurolidine on cell cycle distribution in both the PA-1 andSKOV-3 human ovarian tumor cell lines. The results of these studiesrevealed that exposure to this agent did not induce a consistent patternof cell cycle alterations.

The effect of a 48 h exposure to selected concentrations of Taurolidineon cell cycle distribution in human ovarian tumor cells (PA-1 andSKOV-3) and murine fibroblasts (NIH-3T3) was carried out as follows.Three×10⁵ cells were seeded in plastic tissue culture flasks.Twenty-four hours later, Taurolidine was added to achieve finalconcentrations of 25 μM, 50 μM or 100 μM. Control cultures received anappropriate volume of Kollidine-17P. After an additional 48 h, cellswere harvested, stained with propidium iodide, and cell cycledistribution assessed using cytofluorometric techniques. Each valuerepresents the percentage of cells in the noted cell cycle phases and isthe mean±SEM of three determinations.

TABLE 6 Cell Cycle Distribution, (%) Cell Line/Drug Exposure G₀G₁ S G₂/MNIH-3T3 48 h-0 μM Taurolidine 46.1 ± 9.2 45.0 ± 5.9 9.0 ± 3.3 48 h-25 μMTaurolidine 42.5 ± 9.6 44.9 ± 5.6 13.0 ± 4.0  48 h-50 μM Taurolidine 33.9 ± 10.2 44.3 ± 5.9 21.8 ± 4.6  48 h-100 μM Taurolidine 25.8 ± 1.763.2 ± 9.8 11.0 ± 11.0 PA1 48 h-0 μM Taurolidine 29.9 ± 1.5 47.7 ± 1.022.5 ± 0.5  48 h-25 μM Taurolidine 28.4 ± 0.5 46.8 ± 0.6 24.7 ± 0.9  48h-50 μM Taurolidine 23.7 ± 2.2  39.5 ± 12.5 36.8 ± 12.4 48 h-100 μMTaurolidine 28.4 ± 5.6  44.5 ± 23.5 27.2 ± 17.9 SKOV3 48 h-0 μMTaurolidine 46.7 ± 1.3 38.8 ± 4.1 13.5 ± 3.6  48 h-25 μM Taurolidine45.8 ± 2.7 41.9 ± 4.2 12.3 ± 3.1  48 h-50 μM Taurolidine 30.7 ± 9.4 45.5 ± 12.4 30.3 ± 10.5 48 h-100 μM Taurolidine 19.9 ± 6.1 54.2 ± 8.625.9 ± 7.8 

Specifically, in PA-1 cells, a 48 h exposure to up to 100 μM Taurolidinehad little effect on cell cycle distribution. Indeed, the percentage ofcells in the G₀/G₁-, S-, and G₂/M-phases were essentially unchangeddespite Taurolidine exposure. Alternatively, in SKOV-3 cells,Taurolidine exposure resulted in a concentration-dependent decrease inthe percentage of cells in G₀/G₁ but increased the percentage of cellsin both the S-phase and G₂/M. Importantly, in both the PA-1 and SKOV-3cell lines, Taurolidine exposure also resulted in the appearance of DNAdebris in the sub-G₀G₁ region, an effect that was Taurolidineconcentration-dependent (FIG. 2). Like in the SKOV-3 cell line, exposingNIH-3T3 cells to Taurolidine decreased the percentage of cells in G₀/G₁and increased the percentage of cells in S in a concentration-dependentmanner. However, unlike the human ovarian tumor cells assessed,Taurolidine exposure in NIH-3T3 cells did not significantly affect theappearance of DNA debris in the sub-G₀G₁ region (FIG. 2).

DNA cleavage into discrete fragments is a late event in the process ofapoptosis. The appearance of DNA debris in the sub-G₀/G₁ region 48 hafter Taurolidine exposure could be a reflection of apoptosis-associatedDNA fragmentation. To evaluate this possibility, studies next assessedthe ability of Taurolidine to increase phosphotidylserineexternalization on cell membranes, an event that occurs earlier in theapoptotic process. These studies were fluorocytometry-based and employeda florescent antibody-binding assay (Annexin-V) to assessphosphotidylserine externalization. The results of the studies (shown inFIG. 3) revealed that in both the PA-1 and SKOV-3 human ovarian tumorcell lines a 24 h exposure to Taurolidine induced a significant,Taurolidine-concentration dependent, increase in Annexin-V binding of 4-and 3-fold, respectively. In contrast, in NIH-3T3 cells, Taurolidineexposure resulted in a non-significant increase (˜5%) in antibodybinding. These data supported the results from the cell cycle studies aswell as the observation that Taurolidine exposure induced apoptosis inPA-1 and SKOV-3 cells, but not in NIH-3T3 cells. The results indicatethat Taurolidine preferentially induces apoptosis (and apoptotic death)in tumor cells compared to in non-tumor cells.

To further confirm the induction of apoptosis by Taurolidine, therelationship between Taurolidine exposure and PARP cleavage wasassessed. PARP is a nuclear protein that plays a key role in therecognition and repair of both single and double strand DNA breaks. Inaddition, a key event in the apoptotic process is the cleavage, mediatedby caspase 3 and caspase 9, and consequent catalytic deactivation ofthis protein. To determine if Taurolidine exposure resulted in PARPcleavage in ovarian tumor cells, Western-blot analysis was carried outon whole cell extracts of PA-1, SKOV-3 and NIH-3T3 cells following a 24h exposure to either 50 or 100 μM Taurolidine. The results of thisanalysis, presented in the representative Western-blot contained in FIG.4, revealed that in PA-1 and SKOV-3 cells exposure to either 50 μM or100 μM Taurolidine resulted in PARP cleavage. In contrast, in NIH-3T3cells, following exposure to Taurolidine there was little evidence ofthis proteolytic event. These data confirm that Taurolidine inducesapoptosis in tumor cells but not in non-tumor cells.

Given the preferential induction of apoptotic death in tumor cellscompared to normal nonneoplastic cells, Taurolidine was administered totumor-bearing animals to further evaluate antineoplastic activity.Studies were initiated to evaluate the antineoplastic activity oftaurolidine in nude mice bearing i.p. human ovarian tumor xenografts. Invivo studies were designed to identify the maximally tolerated dose(MTD) regimen of Taurolidine in nude female mice and to assess toxicity.Toxicity was evaluated by measuring changes in body weight, andmortality after a 3 d i.p. bolus injection regimen. Daily 1 mlinjections delivered doses that ranged from 5 mg/mouse/day-30mg/mouse/day. The results of these studies revealed that at daily dosesbelow 15 mg/mouse (˜650 mg/kg) Taurolidine were well-tolerated (Table7).

Taurolidine-induced toxicity in athymic (nude) female mice was evaluatedas follows. Groups of 5-10 mice were injected with Taurolidine on threeconsecutive days. Taurolidine doses evaluated ranged from 5-30mg/mouse/injection and were delivered intraperitoneally in a volume of 1ml (with the exception of the 25 and 30 mg doses, which, due to limitedsolubility, were delivered in a volume of 1.25 and 1.5 ml,respectively). During the injection regimen, and daily thereafter for 30d, mice were weighed and examined. Experiments were repeated a minimumof three times and mortality and weight loss data pooled.

TABLE 7 Taurolidine dose weight loss mortality (mg/mouse/inj) n (nadir%) (%) None (vehicle control) 24  −1.2  0  5 17  −1.2  0 10 17  −1.7  6%15 17  −7.1  0 20 46 −12.2  13% 25 17 −16.3  47% 30 10 −24.5 100%Maximum body weight loss as a result of this dose regimen was 7% andbody weight returned to pre-injection levels within seven days aftercompletion of the injection regimen. With regimens employing doses of 20mg/mouse or greater, more significant toxicity was observed (Table 3).Specifically, nadir weight loss for regimens employing 20, 25 or 30mg/mouse were −12%, −16% and −25%, respectively. Additionally, theseTaurolidine dose regimens resulted in 15%, 43% and 100% mortality,respectively.Based on the toxicity studies, a 3 daily 1 ml i.p. injection ofTaurolidine, at a dose of 20 mg/mouse, was chosen to be the MTD. Studiesnext evaluated the antineoplastic activity of this regimen in micebearing i.p. human ovarian tumor xenografts derived from the SKOV-3 cellline. Mice were injected i.p. with 5×10⁶ SKOV-3 cells. Taurolidinetherapy, employing the 3 d 20 mg/mouse dose regimen, was initiated up to5 d after tumor cell injection. Fourteen days following the terminationof Taurolidine therapy, mice were sacrificed and all i.p. tumors removedand weighed. The results of this study, summarized in FIGS. 5 and 6,revealed that, when initiated on the day of tumor cell injection,Taurolidine therapy was highly effective and inhibited tumor formation(FIG. 5), ascites development, and growth (FIG. 6).

The effect of a single 3 d i.p. bolus injection regimen of Taurolidine(20 mg/mouse/injection, starting on the day of tumor cell injection) onthe gross appearance of mice bearing i.p. xenografts of SKOV-3 humanovarian tumor cells was evaluated. Nineteen days after tumor cellinjection, the mean tumor weight in control mice (no Taurolidine) wasapproximately 1.7 μm. Additionally, control animals were found tocontain up to 7 ml of ascites fluid. Mean tumor weight in thetaurolidine-treated group (single regimen of taurolidine) was less than50 mgs and there was no evidence of ascites formation. A significantnumber of these Taurolidine-treated animals also were found to betumor-free.

When therapy was initiated on the day of tumor cell injection, ˜80% oftreated mice had no evidence of disease upon sacrifice. Further, themean tumor size in treated mice with tumors was approximately 40-foldsmaller and in control (vehicle-treated) mice. Even if Taurolidinetherapy was delayed for up to 3 d after tumor cell injection,approximately 10 percent of mice were tumor free upon sacrifice and themean tumor size in treated mice again was significantly smaller than incontrols. The initiation of this single cycle of Taurolidine therapy 5 dafter tumor cell injection (i.e., in mice with established i.p. ovariantumors) was still capable of significantly inhibiting tumor growth.

The data presented herein indicate that a class of compounds exemplifiedby taurolidine possesses potent antineoplastic activity by selectivelyinhibiting tumor cell growth and specifically induce apoptosis in tumorcells. Surprisingly, the cytotoxic IC₅₀ of Taurolidine was found to bein the 10-50 uM range, approximately 100-fold lower that that requiredfor its antibiotic effects. This difference in effective concentrations,combined with Taurolidine's observed low clinical toxicities indicatesthat this class of compounds is useful as a safe, clinicallywell-tolerated antineoplastic.

The data revealed that exposure to Taurolidine effectively inhibited theproliferation and viability of all tumor cell lines evaluated in a broadpanel of solid tumor cell lines. Taurolidine induced apoptosis inneoplastic cells, indicating that its mechanism of action is not simplyan inhibition of cell surface adhesion components or processes. Resultsof studies carried out in non-adherent cancer cell models support theabove findings and reveal that as little as a 90 min exposure toTaurolidine induces apoptosis in the HL-60 human promyelocytic cellline. Exposure to Taurolidine results in the activation of caspases 3, 8and 9, a disruption of mitochondrial membrane integrity accompanied bycytochrome-C efflux from these organelles, and the cleavage of PARPprotein.

Human leukemia HL-60 cells, which were genetically-engineered to resistapoptotic induction, were induced to apoptosis independently(downstream) of the bcl-2/bax (anti-death gene) point in the signaltransduction cascade. Surprising, in Bcl2-over expressing HL-60 cells,Taurolidine exposure was found to be capable of inducing apoptosis, butwith a delayed onset. These data indicate that an active Taurolidinebreakdown product is capable of reacting with membrane components toaffect intracellular signaling processes and initiate the apoptosisprocess.

The ability of Taurolidine to induce apoptosis was found to be specificfor tumor cells. This observation was confirmed using normal (non-tumor)primary cells derived from animals, which are known to be free oftumors. The cytotoxic and apoptotic activity of Taurolidine in normalmurine bone marrow cultures as well as in activated human T-cellcultures was evaluated. In both normal cell models, Taurolidine was notcytotoxic in the high μM range and did not produce cellular changesconsistent with the induction of apoptosis. In normal murine bonemarrow, concentrations in the mM range were required to inhibit cellproliferation. These findings indicate that Taurolidine (or one of itsmetabolites) access a tumor-cell specific target capable of inducingtumor cell apoptosis.

EXAMPLE 2 Clinical Use

Taurolidine was administered by i.p. lavage immediately followingsurgery for removal of recurrent ovarian tumors. For patients withglioblastoma, Taurolidine was administered systemically. To date,Taurolidine has been well tolerated in these patients.

EXAMPLE 2 Clinical Use

Taurolidine was administered by i.p. lavage immediately followingsurgery for removal of recurrent ovarian tumors. For patients withglioblastoma, taurolidine was administered systemically. To date,Taurolidine has been well tolerated in these patients.

Four patients, which were diagnosed with advanced recurrent glioblastomamultiforma, were treated with taurolidine. The prognosis for this groupof patients was determined to be approximately 8 weeks of survival. Eachpatient received at least one 5 week regimen in which 20 g oftaurolidine was infused intravenously into the arm over a period of 6hours twice a week. In 3 out of the 4 patients treated, the tumor massdecreased or stayed the same; and in one case, a slight increase wasseen. At 14 weeks after the initiation of therapy, each of the patientsremains alive, having exceeded the 8 week prognosis. A beneficialclinical effect was achieved in these brain tumor patients with systemicadministration of taurolidine, indicating that taurolidine, or ametabolite of taurolidine, successfully crossed the blood-brain barrierto gain access to the tumor in the brain.

These data indicate that taurolidine is useful to inhibit or halt tumorgrowth and to extend the life expectancy of tumor patients

Other embodiments are within the following claims.

1. A method of inducing apoptotic death of a neoplastic cell, comprisingcontacting said cell with an apoptosis-inducing amount of amethylol-containing compound, wherein said neoplastic cell is acarcinoma cell and wherein said compound is taurolidine or a derivativethereof and wherein said neoplastic cell is a metastasis from a non-CNSprimary tumor.
 2. A method of inducing apoptotic death of a neoplasticcell, comprising contacting said cell with an apoptosis-inducing amountof a methylol-containing compound, wherein said neoplastic cell is acarcinoma cell and wherein said compound is taurolidine or a derivativethereof and wherein said neoplastic cell is a multiple drug resistanttumor cell.
 3. A method of inducing apoptotic death of a neoplasticcell, comprising contacting said cell with an apoptosis-inducing amountof a methylol-containing compound, wherein said neoplastic cell is acarcinoma cell and wherein said compound is taurolidine or a derivativeand wherein said neoplastic cell is an ovarian carcinoma cell.